Collagen Membranes Adsorb the Transforming Growth Factor‐β Receptor I Kinase‐Dependent Activity of Enamel Matrix Derivative

Background: Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)‐β signaling in oral fibroblasts. Methods: Three commercially available collagen products were exposed to EMD or recombinant TGF‐β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF‐β target genes interleukin (IL)‐11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen. Results: EMD or TGF‐β1 provoked a significant increase of IL‐11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF‐β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin‐coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL‐11 and PRG4. Conclusion: These in vitro findings suggest that collagen products adsorb a TGF‐β receptor I kinase‐dependent activity of EMD and make it available for potential target cells.     

Quantitative Analysis of Interdental Gingiva in Patients With Chronic Periodontitis and Transforming Growth Factor‐β1 29C/T Gene Polymorphisms

Background: The association of transforming growth factor (TGF)‐β1 29C/T gene polymorphisms with level of tissue breakdown and periodontal disease progression is not clear. In this study, quantitative parameters of interdental papilla are investigated in patients with chronic periodontitis (CP) and TGF‐β1 29C/T gene polymorphisms. Methods: Sixty gingiva samples were included. After determination of TGF‐β1 29C/T gene polymorphisms using tetra‐primer amplification refractory mutation system/polymerase chain reaction (T‐ARMS‐PCR), 15 gingival tissue samples from patients with CP in each genotype (TT, TC, and CC) were considered as case groups. Fifteen control samples were also collected from healthy individuals. After tissue processing, interdental gingiva tissues were exhaustively sectioned into 4‐μm‐thick sections. Ten to 13 sections were sampled by systematic uniform random sampling and stained with Masson trichrome, and the volume density (V_v) of the gingival components was estimated using Cavalieri estimation. Results: Statistically significant differences were found in V_v of epithelium, connective tissue, collagenous and non‐collagenous matrix, and blood vessels between control and CP groups (P <0.0001). There was a corresponding decrease in the collagenous matrix V_v in patients with the TT genotype compared with those with CT and CC genotypes. Collagenous matrix and blood vessel V_v values were statistically correlated with the number of T alleles (r = ?0.74, r² = 54.8%, P = 0.0001 and r = 0.84, r² = 70.6%, P = 0.0001, respectively). Conclusion: This study shows that there is a strong association between TGF‐β1 29C/T gene polymorphisms and quantitative parameters of interdental papilla in patients with CP.     

Tumor Necrosis Factor‐α and Porphyromonas gingivalis Lipopolysaccharides Decrease Periostin in Human Periodontal Ligament Fibroblasts

Background: Periostin is a matricellular protein essential for tissue integrity and maturation and is believed to have a key function as a modulator of periodontal ligament (PDL) homeostasis. The aim of this study is to evaluate whether periodontal disease‐associated pathogen‐related virulence factors (endotoxins/lipopolysaccharides [LPS]) and proinflammatory cytokines alter the expression of periostin in PDL cells. Methods: Human PDL cultures were exposed to inflammatory mediators (tumor necrosis factor‐α [TNF‐α]), bacterial virulence factors (Porphyromonas gingivalis LPS) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. Periostin and TGF‐β inducible gene clone H3 (βIGH3) mRNA expression from cell lysates were analyzed. Periostin and βIGH3 proteins were also detected and semiquantified in both cell lysates and cell culture supernatants by Western blot. In addition, periostin localization by immunofluorescence was performed. Analysis of variance and Fisher tests were used to define the statistical differences among groups (P <0.05). Results: In a mechanically challenged environment, periostin protein was more efficiently incorporated into the matrix compared to the non‐loaded controls (higher levels of periostin in the supernatant in the non‐loaded group). Interestingly, chronic exposure to proinflammatory cytokines and/or microbial virulence factors significantly decreased periostin protein levels in the loaded cultures. There was greater variability on βIGH3 levels, and no particular pattern was clearly evident. Conclusions: Inflammatory mediators (TNF‐α) and bacterial virulence factors (P. gingivalis LPS) decrease periostin expression in human PDL fibroblasts. These results support a potential mechanism by which periostin alterations could act as a contributing factor during periodontal disease progression.     

Role of Transforming Growth Factor‐beta1 in Cyclosporine‐Induced Epithelial‐to‐Mesenchymal Transition in Gingival Epithelium

Background: It has been proposed that cyclosporin A (CsA) may induce epithelial‐to‐mesenchymal transition (EMT) in gingiva. The aims of the present study are to confirm the notion that EMT occurs in human gingival epithelial (hGE) cells after CsA treatment and to investigate the role of transforming growth factor beta1 (TGF‐β1) on this CsA‐induced EMT. Methods: The effects of CsA, with and without TGF‐β1 inhibitor, on the morphologic changes of primary culture of hGE cells were examined in vitro. The changes of protein and messenger RNA (mRNA) expressions of two EMT markers (E‐cadherin and alpha‐smooth muscle actin) in the hGE cells after CsA treatment with and without TGF‐β1 inhibitor were evaluated with immunocytochemistry and real‐time polymerase chain reaction. Results: The epithelial cells became spindle‐like, elongated, and disassociated from neighboring cells and lost their original cobblestone monolayer pattern when CsA was added. However, the epithelial cells stayed in their original cobblestone morphology with treatment of TGF‐β1 inhibitor on top of the CsA treatment. When CsA was given, the protein and mRNA expressions of E‐cadherin and α‐SMA were significantly altered, and these alterations were significantly reversed with pretreatment of TGF‐β1 inhibitor. Conclusions: CsA could induce Type 2 EMT in gingiva by changing the morphology of epithelial cells and altering the EMT markers/effectors. The CsA‐induced gingival EMT is dependent or at least partially dependent on TGF‐β1.     

Orthodontic Force Increases Interleukin‐1β and Tumor Necrosis Factor‐α Expression and Alveolar Bone Loss in Periodontitis

Background: The present study aims to evaluate the effects of orthodontic movement (OM) on the periodontal tissues of rats with ligature‐induced periodontal disease. Methods: Eighty‐eight rats were divided into four groups: 1) negative control (sham operated); 2) periodontal disease; 3) OM; and 4) periodontal disease followed by OM (OMP). Rats were sacrificed 3 hours or 1, 3, or 7 days after OM commencement. Bone volume fraction (BVF) and bone mineral density (BMD) were assessed in hemimaxillae by microcomputed tomography analysis. Expression of the proinflammatory cytokines interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α were evaluated in gingival samples by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, and in the furcation region by immunohistochemistry analysis (IHC). Results: The OMP group had lower BVF and BMD levels compared to the other groups at day 7 (P <0.05). Maximum messenger ribonucleic acid expression of both cytokines was observed in the OMP group at day 1 (P <0.05). In the same period, all proteins were expressed in high levels for all test groups compared to the control group. The number of cells positive for IL‐1β and TNF‐α by IHC was highest in the OMP group at day 1, with progressive reduction thereafter. Conclusion: The results suggest that OM acts synergistically with periodontal disease in periodontal breakdown through upregulation of proinflammatory cytokines.     

Tumor Necrosis Factor‐α Induces Matrix Metalloproteinases‐3, ‐10, and ‐13 in Human Periodontal Ligament Cells

Background: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)‐α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus. Methods: Cultured PDL cells were stimulated with TNF‐α and analyzed with an MMP antibody array. Real‐time polymerase chain reaction (PCR), enzyme‐linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP‐3, ‐10, and ‐13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP‐3, ‐10, and ‐13 was observed after blocking the TNFR with an antagonist. Results from real‐time PCR, ELISA, and western blot were analyzed by paired t test. Results: The antibody array showed that the protein most strongly upregulated by TNF‐α stimulation was MMP‐3, followed by MMP‐13 and MMP‐10. The TNF‐α receptor blocker specifically inhibited expression of MMP‐3 and ‐13. In addition, TNF‐α increased levels of MMP mRNAs in MMP‐3, ‐13, and ‐10 (in decreasing order). However, ELISAs showed that MMP‐13 was the most upregulated protein, followed by MMP‐10 and MMP‐3. Western blotting indicated that TNF‐α increased MMP‐3 and ‐13 levels but had no significant effect on the level of MMP‐10, and zymography showed that TNF‐α increased the activities of all forms of MMP‐3 and ‐13, but MMP‐10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1. Conclusions: TNF‐α (10 ng/mL) upregulates levels of MMP‐3, ‐10, and ‐13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.     

Periodontal Condition of Patients With Autoimmune Diseases and the Effect of Anti‐Tumor Necrosis Factor‐α Therapy

Background: The aim of this study is to evaluate the effect of autoimmune diseases (AIs), as well as anti‐tumor necrosis factor‐α (TNF‐α) therapy on the clinical and immunologic parameters of the periodontium. Methods: Thirty‐six AI patients (12 rheumatoid arthritis [RA], 12 psoriatic arthritis, and 12 systemic sclerosis) were recruited together with 12 healthy (H) and 10 RA patients receiving anti‐TNF‐α therapy (RA+). Periodontal indices including plaque index, gingival index (GI), probing depth (PD), and bleeding on probing (BOP) were measured, and gingival crevicular fluid (GCF) was collected from five deepest pockets using papers strips. The TNF‐α level was analyzed using enzyme‐linked immunosorbent assay. Analysis of variance test was used for statistical comparison between groups, whereas Pearson linear correlation coefficient test was used to examine the association between TNF‐α and periodontal status indices. Results: The three AI subgroups were very similar in clinical and immunologic parameters. GI was greater in the AI patients compared to the H and RA+ groups (1.91 ± 0.54, 1.21 ± 0.67, and 1.45 ± 0.30, respectively, P = 0.0005). AI patients exhibited significantly more BOP than H and RA+ (46.45% ± 17.08%, 30.08% ± 16.86%, and 21.13% ± 9.51%, respectively, P = 0.0002). PD in H and RA+ groups were lower than in the AI (3.47 ± 0.33, 3.22 ± 0.41, and 3.91 ± 0.49 mm, P = 0.0001). Number of sites with PD >4 mm was higher in AI patients compared to H and RA+ (42.44 ± 17.5 versus 24.33 ± 15.62 versus 33.3 ± 6.6, P = 0.0002). GCF TNF‐α was higher among the AI patients (1.67 ± 0.58 ng/site) compared to 1.07 ± 0.33 ng/site for the H group and 0.97 ± 0.52 ng/site for the RA+ group (P = 0.0002). A significant positive correlation was found between PD and TNF‐α levels in the GCF (r = 0.4672, P = 0.0002), BOP (r = 0.7491, P = 0.0001), and GI (r = 0.5420, P = 0.0001). Conclusions: Patients with AI diseases have higher periodontal indices and higher TNF‐α levels in GCF than H controls. Anti‐TNF‐α treatment appears to reverse this phenomenon.     

Effect of Intracanal Medicaments and Irrigants on the Release of Transforming Growth Factor Beta 1 and Vascular Endothelial Growth Factor from Cervical Root Dentin

IntroductionThis study aimed to evaluate (1) the effect of irrigating solutions and intracanal medicaments on the release of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF) from cervical root dentin and (2) the effect of associating triple antibiotic paste (TAP) and calcium hydroxide paste (CH) with 2% chlorhexidine (CHX) on TGF-β1 release.MethodsFirst, 119 specimens from roots (cervical thirds) were obtained and were distributed into 5 groups: 2% CHX, 2.5% sodium hypochlorite, TAP, CH, and 10% EDTA by each growth factor (TGF-β1 [n = 8] and VEGF [n = 8]). Then, specimens were distributed as follows (n = 13): TAP + 2% CHX, CH + 2% CHX, and 10% EDTA and treated with irrigating solutions and intracanal medicaments. After the treatments, the specimens were immersed in 10% EDTA (20 minutes), and the solution was analyzed using the enzyme-linked immunosorbent assay. The data were submitted to normality, homogeneity of variance, and Mann-Whitney tests (P < .05).ResultsSignificant differences were found between the irrigating solutions (P < .05) and intracanal medicaments for TGF-β1 (P < .05). No VEGF release was detected for any group. Our results showed no significant differences among the TAP + 2% CHX and EDTA groups for TGF-β1 but a significant difference between CH + 2% CHX and the other groups (P < .05).ConclusionsThe use of 2% CHX as the irrigating solution, CH as the intracanal medicament, and 10% EDTA as the final irrigation provides higher TGF-β1 release from the cervical root dentin, whereas VEGF was not detected. Moreover, TAP and 2% CHX with 10% EDTA as the final irrigation resulted in greater TGF-β1 release from cervical root dentin than CH + 2% CHX.     

The Effect of Transforming Growth Factor Beta 1 on the Mineralization of Human Cementoblasts

IntroductionTransforming growth factor beta 1 (TGF-β1) plays an important role in bone mineralization and has been reported to promote osteoblast proliferation and differentiation. However, there is no report about the effects of TGF-β1 on human cementoblasts. The purpose of this study was to clarify the effect of TGF-β1 on the proliferation and differentiation of the human cementoblast cell line (HCEM) in vitro.MethodsHCEM cells were stimulated with TGF-β1 at concentrations of 0.05, 0.5, 5, and 10 ng/mL. A proliferation assay was performed from 24–72 hours. The effect of TGF-β1 on mineralization was analyzed by quantifying the area stained with alizarin red on days 7 and 14. Real-time polymerase chain reaction was used to assess the effect of TGF-β1 on the mineralization-related genes alkaline phosphatase, bone sialoprotein, and type I collagen on days 3, 7, and 14.ResultsTGF-β1 did not affect cell proliferation. TGF-β1 together with the mineralization medium (consisting of ascorbic acid, dexamethasone, and β-glycerophosphate) increased the alizarin red–stained area on days 7 and 14. Real-time polymerase chain reaction revealed that alkaline phosphatase messenger RNA expression was increased in TGF-β1–stimulated HCEM cells in mineralization medium on days 3 and 7, whereas bone sialoprotein and type I collagen messenger RNA expression was increased on day 7.ConclusionsAlthough TGF-β1 does not affect cell proliferation, it does promote cell differentiation and mineralization of HCEM cells.     

Tumor Necrosis Factor‐α and Oral Inflammation in Patients With Crohn Disease

Background: Crohn disease (CD) is a chronic inflammatory bowel disease often accompanied by periodontal symptoms. Based on its function in immune response, tumor necrosis factor (TNF)‐α and its genetic variants have been discussed as risk indicators in inflammatory processes. Therefore, the aim of the present study is to investigate the impact of TNF‐α polymorphisms on periodontal parameters and inflammatory lesions of oral mucosa as a characteristic of CD. Methods: A total of 142 patients with CD were included in the study. Oral soft tissue alterations and periodontal parameters were assessed. Genotypes, alleles, and haplotypes of TNF‐α polymorphisms (rs1800629, cDNA?308G > A; and rs361525, cDNA?238G > A) were determined by polymerase chain reaction with sequence‐specific primers (PCR‐SSP). Results: Patients with CD who exhibit more severe oral soft tissue alterations were significantly more often A allele carriers of rs361525 than G allele carriers (14.2% versus 2.2%; P <0.001). Furthermore, A allele carriers had a higher mean periodontal probing depth (P <0.05), mean clinical attachment level (P <0.05), and sites with bleeding on probing (not significant). Similar results were obtained when evaluating A allele‐containing genotypes (AG + AA) and haplotypes (GA). In multivariate analyses considering age, sex, smoking, and medication as confounders, the A allele was proven to be an independent risk indicator for oral soft tissue alterations in patients with CD. No genotype‐dependent influence of rs1800629 was observed. Conclusion: The TNF‐α A allele of rs361525 represents a significant risk indicator for oral soft tissue alterations in patients with CD.     

Cyclosporine A Enhances Gingival β‐Catenin Stability via Wnt Signaling

Background : Cyclosporine A (CsA) increases β‐catenin messenger RNA (mRNA) and protein expression. The present study demonstrates that Wnt/β‐catenin signaling inhibits β‐catenin degradation in the gingiva. Methods: Forty 5‐week‐old male Sprague‐Dawley rats were assigned to two study groups after healing from right maxillary molar extractions. The rats in the experimental group were fed 30 mg/kg CsA daily for 4 weeks, whereas the control rats were fed mineral oil. At the end of the study, all rats were sacrificed, and the gingivae were obtained. The gingival morphology after CsA treatment was evaluated by histology, and the genes related to Wnt/β‐catenin signaling were initially screened by microarray. Polymerase chain reaction, Western blotting, and immunohistochemistry were used to examine the mRNA and protein expression of proliferating cell nuclear antigen, cyclin D1, E‐cadherin, β‐catenin, Dvl‐1, glycogen synthase kinase‐3β, axin‐1, and adenomatous polyposis coli (APC). Phosphoserine and ubiquitinylated β‐catenin were detected after immunoprecipitation. Results: In rats treated with CsA, overgrowth of gingivae was observed, and altered expression of genes related to Wnt/β‐catenin signaling was detected by the microarray. The gingival mRNA and protein expression profiles for genes associated with Wnt/β‐catenin signaling further confirmed the effect of CsA: β‐catenin and Dvl‐1 expression increased, but APC and axin‐1 expression decreased. Western blotting and immunohistochemistry showed decreases in β‐catenin serine phosphorylation (33/37) and ubiquitinylation in the gingivae of CsA‐treated rats. Conclusion: CsA‐enhanced gingival β‐catenin stability may be involved in gene upregulation or β‐catenin degradation via the Wnt/β‐catenin pathway.